PLRV and PVY are the two most important virus diseases of potato worldwide, responsible for serious yield losses and degeneration of vegetative seed. PVX, although less important alone, is commonly found in combined infections with other viruses, interactions among which increase the disease effect. PLRV and PVY are transmitted by aphids, while PVX and PVY are also transmitted by contact. This standard evaluation protocol serves to assess the reaction of potato clones to viruses, through their intentional exposure to viruses at mid to late stages of selection programs.

1. Background

Until now, three different types of resistance to PVX and PVY have been recognized: extreme resistance, relative resistance, and hypersensitivity. The former confers the highest level of resistance, and is the most stable with respect to pathogen variants and environmental conditions. Clones with extreme resistance inhibit virus multiplication to such an extent that no virus can be recovered from plants by indexation. On the other hand, relative resistance, which is polygenic in nature, confers varying degrees of field resistance to virus infection. Hypersensitivity or ‘field immunity’ is a variant-specific resistance expressed as a local necrotic reaction of infected cultivars, which localizes the virus and hampers its spread within the plant. Extreme resistance and hypersensitivity can be easily assessed in a sample of few plants of a cultivar by mechanical and graft inoculation of the virus under greenhouse conditions, while relative resistance requires at least three seasons of exposure to high inoculum pressure.

Relative resistance is the main and most common type of resistance encountered against PLRV. Commonly deployed sources are of a polygenic nature, influenced by environmental factors such as vector and inoculum pressure. As PLRV is not mechanically transmissible, test clones must be exposed to it via either vectors or grafting. Two components of resistance to PLRV have been identified: resistance to infection and resistance to multiplication.  Infection rates depend on the level of susceptibility of the clone and the vector pressure encountered during the growing season. Evaluation of this type of resistance has traditionally been carried out by field exposure trials in locations selected for their medium to high levels of aphid pressure. Assessment is based on the percentage of plants that become infected in a given number of exposure cycles. Clones with less than 35% of infected plants after at least three exposure seasons are considered resistant to infection. The present protocol describes intentional exposure trials for assessment of resistance to these three viruses in advanced selection programs. A practical methodology to assess both extreme resistance and hypersensitivity to PVX and PVY is also described.

2. Methodology

The first step in evaluating promising advanced clones for their reaction to viruses is to carefully observe plants for symptoms in selection plots, and conduct ELISA serology to confirm infection by a given virus. PVX- and PVY-infected clones may be identified by careful inspection of hill plots, recording those showing mosaic (Fig 1) and/or systemic necrosis (Fig 2) symptoms. Observations are conducted 45-50 days after planting for early maturing clones (90 days) and 55-60 days for late ones (120 or more days), and should be carried out early in the morning or under over-cast skies for better visualization of symptoms. Infection by PVX and PVY in these symptomatic plants should be confirmed by ELISA serology. A sample of two or three leaflets showing mosaic are taken from the symptomatic plant, and put in a small polyethylene plastic bag and sent to the laboratory. A positive result in ELISA confirms infection and the lack of extreme resistance, while a negative result does not necessarily mean that the clone has extreme resistance to the virus in question. Rouguing of infected plants is not recommended, to permit continued evaluation of relative resistance in subsequent growing cycles.

Figure 1. Mosaic symptoms induced by PVX or PVY

Figure 2. Necrotic symptoms induced by PVX or PVY
2.1. Field Exposure Trials

Field exposure trials involve three seasons of intentional field exposures to viruses. PVY and PLRV susceptible cultivars, and one resistant to PLRV should be included as controls in every trial. . Field disease trials are carried out by planting test material in between infector plants. Infector plants are PLRV infected plants , obtained from tubers of infected plants of a PLRV susceptible cultivar grown under natural field infection for at least two seasons in the same location, before beginning Field Disease Trials. Enough tubers from infected plants should be available to carry out these trials. We use two susceptible local cultivars as infectors: "Ticahuasi" and " Perricholi". Tubers of advanced clones to be tested should come from multiplication or agronomic selection trials carried out in an area with no or very low aphid pressure. After the third exposure trial under a moderate aphid pressure area, those advanced clones with less than 35% of PLRV infection can be considered resistant. In the case of PVX and PVY, clones with 20% or fewer plants infected can be considered resistant. Those advanced clones that have not shown infection with any of these two latter viruses, may undergo graft and mechanical inoculation to determine other types of resistance i.e., extreme resistance or hypersensitivity.

2.1.1. First Exposure Trial

Test clones should be planted in between infectors as shown in Figure 3. Ten tubers of each clone should be available to begin this trial. Infectors are planted two weeks before the test material in order to permit their colonization before the test material, and thus allow aphids to acquire inoculum.  Test clones are planted in ten hill-plots without replications.

Aphids are counted forty days after infectors were planted. A random sample of 5 infector rows are taken and aphids are counted on each plant. A mean number between 15 to 20 aphids/plant indicates a moderate aphid pressure. If less than a mean number of 10 aphids/plant are found, it is recommended to increase the population by setting "Chinese cabbage" (Brassica perkinensis) or  virus-free Datura stramonium  leaves infested with aphids on the infector plants.

Aphids are counted again 15 days after the first count, but this time counting is carried out on a random sample of 5 test rows. If a mean number of 40 aphids/plant or more are found, an application of a contact pesticide (i.e, Pirimor 3 º/oo) is recommended to moderate exposure.

Plant evaluation or ELISA for PLRV infection is not necessary or reliable in this exposure trial, as plants that acquire infection for the first time (primary infection) or are infected  late during the growing season may look healthy and give false negative reactions. However, ELISA can be performed in leaf samples of the ten plants of the susceptible control and in a random sample of three test clones, to verify infection. The test can be performed 50 days after planting. At this time, evaluation for PVX and PVY infection is also performed as described before. Virus infection on symptomatic plants is confirmed by ELISA. The number of infected plants/clone is recorded in the notebook. At harvest, two tubers from each plant of every test clone are recovered and put in a net bag labeled with the clone name. Thus, each test clone bag will have 20 tubers that will be used for planting  the second exposure trial.

2.1.2 Second Exposure Trial

This trial is conducted in RCBD with two replications of ten hill-plots. Infectors are planted in between test rows as described before. Controls are also included, using tubers recovered from the first exposure trial. Aphid counting is done as described for the first exposure trial, as well as decisions to increase or decrease the number of aphids. Evaluation of PVX and PVY infection is carried out on each plant 50-55 days after planting. Symptomatic plants are confirmed by ELISA. The number of infected plants per clone and per block is registered in the notebook. At this time, PLRV symptom evaluation is also carried out on individual plants. ELISA is conducted on leaf samples of plants in those cases where doubts or discrepancies between scorers arise, or where no symptoms (asymptomatic plants) are observed. Rolling of lower leaves and/ or chlorosis of apical leaves are typical symptoms of secondary infection with PLRV (Fig 4). Secondary symptoms arise from plants of infected tubers. The number of infected plants should be registered per clone and per replication, and the mean percentage of infection of each clone estimated from the two replications. After this exposure trial, advanced clones with more than 50 % infection by any of the viruses tested must be declared susceptible to the virus in question.

Figure 4. Chlorosis and Leafrolling induced by PLRV.
2.1.3. Third Exposure Trial

For this trial, 30 tubers harvested from the second exposure trial are used. The trial is carried out in a RCBD with three replications of ten hill-plots. If the susceptible control reached 80% or more infection in the second exposure trial, and aphid pressure was moderate to high, there is no need to use infector plants, and aphid counting is done only to control overpopulation. Virus symptom evaluation and ELISA test is performed on individual plants 50-55 days after planting. Percentages of PLRV, PVX and PVY infection are independently estimated as the number of infected plants (positive in ELISA) over the total number of plants tested per clone and replication. Mean percentage of infection is obtained from the three replications.

At this stage, it is possible to measure the degenerative effect of virus infection on yield, by planting a similar trial close to this one, using tubers harvested from trials conducted for at least two seasons under no or very low aphid pressure (i.e., with no or low virus content). A combined analysis of the two trials can be performed to obtain the error mean square necessary to carry out statistical comparison between yields of each clone under presence and absence of virus infection pressure in the previous growing seasons.

Those advanced clones with 35% or fewer plants infected may be considered resistant to PLRV infection. In the case of PVX and PVY, those with 20% or less infection are considered to possess relative resistance to these viruses. Those clones that do not suffer yield reduction despite high rates of virus infection are considered tolerant. Clones that have not shown infection to PVX, PVY, or both may undergo graft and mechanical inoculation to determine the type of resistance present.

2.2. Greenhouse trials to assess extreme resistance and hypersensitivity to PVX and PVY

A set of seven plants per clone is used to test for resistance to each virus. Plants can be obtained from tubers, cuttings or sprouts, and a PVX and PVY susceptible potato cultivar should be included as control.

Materials
  1. Test clones.
  2. PVX infected young plants of Nicotiana glutinosa (twenty infected plants are enough for inoculation of 100 plants).
  3. PVX infected plants of a PVX susceptible cultivar (to provide infected scions for graft inoculation)
  4. Young PVY infected plants of Nicotiana occidentalis (twenty infected plants are enough for inoculation of 100 plants).
  5. Healthy young plants of Nicotiana glutinosa and Nicotiana occidentalis for indexation (two plants per test clone)
  6. Small plastic bags to take samples for ELISA test.
  7. ELISA kit.   
  8. Pencil and notebook.
2.2.1 Mechanical and graft inoculation

This test should be carried out during seasons with temperatures between 16o and 25o C. Inoculations are carried out using the PVYo strain of PVY, and the PVX strain member of Cockerham's group 2 of PVX strains, which induces severe mosaic and necrosis. These strains are maintained by continous mechanical inoculations in plants of Nicotiana. occidentalis and N. glutinosa, respectively. However, you may use a local virus isolate.

Three plants of each set are mechanically inoculated with PVX and PVY, respectively, 15 days after planting, i.e., when plants are still young or about 10 cm in height. Inoculum is prepared as a suspension of PVX-infected leaves of N.glutinosa, and PVY-infected leaves of N. occidentalis, in cold distilled water 5% w/v.  Leaves of the test clones are dusted with 600 mesh carborundum and inoculation is carried out by rubbing two or three leaflets of each plant with a piece of cheesecloth previously dipped in the inoculum (Fig 5).

Three additional plants in each set are graft-inoculated 25 days after planting, or when plants are 25 cm in height. Inoculation is carried out by grafting scions of infected source plants onto individual potato plants (Fig 6). Scions are obtained from PVY-infected plants of N. occidentalis, and from infected plants of any PVX susceptiblepotato cultivar that develops only mosaic symptoms. (Cultivars that develop necrotic symptoms are not desirable since they transmit the virus less successfully; for this reason the cultivar Rosita is used at CIP). The remaining (healthy control) plant of each set is kept in the same greenhouse, but not in contact with the inoculated plants, for purposes of observing reactions of the clone to virus or other factors. Scions are removed from plants 20 days after grafting to avoid any passive translocation of the virus that could be detected by ELISA.

Figure 5. Mechanical inoculation of test clones

Figure 6. Graft inoculation of test clones (PVY-infected scion of N. occidentalis)
2.2.2. Evaluation

Evaluations are recommended 25 days after mechanical or graft inoculation, although symptoms on the susceptible controls are the best indicators of the appropriate evaluation time.

i) Visual symptoms

Plants of each clone are individually assessed for mosaic (M), systemic necrosis (SN), and local necrotic symptoms (LN) . Local necrosis is recognized by tiny necrotic spots on inoculated leaves or dropping of the inoculated leaf. However depending on environmental conditions, local necrosis may develop into systemic necrosis (Fig 2). This highly hypersensitive reaction should be considered as extreme susceptibility, since plants tend to either self eliminate, or develop mosaic symptoms.

ii) ELISA

ELISA is performed on composite samples of leaves of plants inoculated with the same virus and inoculation technique i.e., a sample of leaves from 3 plants mechanically inoculated with PVX.  Clones that develop systemic necrosis are characterized as susceptible, even though the virus is not detected by indexing, since they without doubt will develop mosaics in future generations.

2.2.3 Back testing on indicator plants

Those clones that result negative in ELISA are indexed on virus-free indicator plants of N. occidentalis or N. glutinosa for PVY or PVX detection, respectively. A mixed sample of leaflets taken from all plants of the putative resistant clone is crushed in cold distilled water (1:5) inside a small plastic bag (Fig 7). A set of two young plants of the respective indicator for each virus, is dusted with 600 mesh carborundum, and inoculated (Fig 8). The indicator plants are labeled with the code or name of the test clone, and assessed for mosaic symptoms 15 days after inoculation. (An ELISA test can be carried out if desired to confirm virus presence) If none of the indicator plants shows mosaic symptoms, the respective test clone can be declared an extreme resistant one, unless local necrosis had been observed in the clone, in which case the clone is recorded as hypersensitive.

Figure 7. Mechanical inoculation of test clones

Figure 8. Graft inoculation of test clones (PVY-infected scion of N. occidentalis)
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